Treatment of tumors using specific anti-L1 antibody

ABSTRACT

The present invention relates to the anti-L1 monoclonal antibody 9.3 as well as to related antibodies or binding molecules and well as to the uses thereof, especially in tumor treatment.

This application claims benefit of U.S. provisional application Ser. No.60/944,359 filed Jun. 15, 2007, which is incorporated herein byreference in its entirety.

The present invention relates to the treatment of tumors by the use of aspecific anti-L1 antibody.

The standard treatment of advanced cancer is often chemotherapy orradiotherapy. However, despite initial response to therapy, it is oftenobserved that different carcinomas acquire resistance tochemotherapeutic drugs or radiotherapy leading to tumor recurrence andfrequent death of the patients. Often, it is then decided to switch toanother chemotherapeutic drug or to higher dosages. However, often noimprovement of the clinical situation is observed.

L1 is a type I membrane glycoprotein of 200 to 230 kDa structurallybelonging to the Ig superfamily (Moos M, Tacke R, Scherer H, Teplow D,Fruh K, Schachner M. Neural adhesion molecule L1 as a member of theimmunoglobulin superfamily with binding domains similar to fibronectin.Nature 1988; 334:701-3). L1 plays a crucial role in axon guidance andcell migration in developing nervous system (Hortsch M. Structural andfunctional evolution of the L1 family: are four adhesion moleculesbetter than one? Mol Cell Neurosci 2000; 15:1-10., Schachner M. Neuralrecognition molecules and synaptic plasticity. Curr Opin Cell Biol 1997;9:627-34). Recent studies have also implicated L1 expression in theprogression of human carcinomas. L1 expression was found on differenttumors including lung cancer (Katayama M, Iwamatsu A, Masutani H, FurukeK, Takeda K, Wada H, et al. Expression of neural cell adhesion moleculeL1 in human lung cancer cell lines. Cell Struct Funct 1997; 22:511-6),gliomas (Senner V, Kismann E, Puttmann S, Hoess N, Baur I, Paulus W. L1expressed by glioma cells promotes adhesion but not migration. Glia2002; 38:146-54), melanomas (Thies A, Schachner M, Moll I, Berger J,Schulze H J, Brunner G, et al. Overexpression of the cell adhesionmolecule L1 is associated with metastasis in cutaneous malignantmelanoma. Eur J Cancer 2002; 38:1708-1, Fogel M, Mechtersheimer S,Huszar M, Smirnov A, Abu D A, Tilgen W, et al. L1 adhesion molecule (CD171) in development and progression of human malignant melanoma. CancerLett 2003; 189:237-47), renal carcinoma (Meli M L, Carrel F, Waibel R,Amstutz H, Crompton N, Jaussi R, Moch H, Schubiger P A, Novak-Hofer I.Anti-neuroblastoma antibody chCE7 binds to an isoform of L1-CAM presentin renal carcinoma cells. Int J Cancer, 1999; 83: 401-408, Allory Y,Matsuoka Y, Bazille C, Christensen El, Ronco P, Debiec H. The L1 celladhesion molecule is induced in renal cancer cells and correlates withmetastasis in clear cell carcinomas. Clin Cancer Res 2005; 11:1190-7)and colon carcinoma (Gavert N, Conacci-Sorrell M, Cast D, Schneider A,Altevogt P, Brabletz T, et al. L1, a novel target of beta-cateninsignaling, transforms cells and is expressed at the invasive front ofcolon cancers. J Cell Biol 2005; 168:633-42). Furthermore, it is knownin the art that L1 is overexpressed in ovarian and endometrialcarcinomas in a stage-dependent manner (Fogel M, Gutwein P,Mechtersheimer S, Riedle S, Stoeck A, Smirnov A, et al. L1 expression asa predictor of progression and survival in patients with uterine andovarian carcinomas. Lancet 2003; 362:869-75).

In the art, it has been suggested to use anti-L1 antibodies for thetreatment of ovarian and endometrial tumors (cf. WO 02/04952, WO06/013051 and Arlt M J, Novak-Hofer I, Gast D, Gschwend V, MoldenhauerG, Grunberg J, et al. Efficient inhibition of intra-peritoneal tumorgrowth and dissemination of human ovarian carcinoma cells in nude miceby anti-L1-cell adhesion molecule monoclonal antibody treatment. CancerRes 2006; 66:936-43). In the art, various anti-L1 antibodies are known(e.g. mAb 14.10: Huszar M, Moldenhauer G, Gschwend V, Ben-Arie A,Altevogt P, Fogel M: Expression profile analysis in multiple humantumors identifies L1 (CD171) as a molecular marker for differentialdiagnosis and targeted therapy. Hum Pathol 37:1000-1008, 2006, mabchCE7: Meli M L, Carrel F, Waibel R, Amstutz H, Crompton N, Jaussi R,Moch H, Schubiger P A, Novak-Hofer I: Anti-neuroblastoma antibody chCE7binds to an isoform of L-CAM present in renal carcinoma cells. Int JCancer 83:401-408, 1999, mAb UJ127.11: Patel K, Kiely F, Phimister E,Melino G, Rathjen F, Kemshead J T: The 200/220 kDa antigen recognized bymonoclonal antibody (MAb) UJ127.11 on neural tissues and tumors is thehuman L1 adhesion molecule. Hybridoma 10:481-491, 1991, mAb 5G3: Wolff JM, Frank R, Mujoo K, Spiro R C, Reisfeld R A, Rathjen F G: A human brainglycoprotein related to the mouse cell adhesion molecule L1. J Biol Chem263:11943-11947, 1988). Furthermore, in Sebens Müerkoster et al.,Oncogene. 2007 Apr. 26; 26(19):2759-68, Epub 2006 Nov. 6, it has beensuggested to use anti-L1 antibodies for sensitizing tumor cells for thetreatment with a chemotherapeutic drug or with radiotherapy.

There is always a need for improved anti-tumor agents.

The present invention relates in one aspect to an anti-L1 monoclonalantibody which is capable of binding to the same L1 epitope recognizedby the monoclonal antibody 9.3, produced by the hybridoma cell depositedunder DSMZ ACC2841.

In the context of the present invention, it has been surprisingly foundthat the monoclonal antibody 9.3, produced by the hybridoma celldeposited under DSMZ ACC2841, has improved anti-tumor capacities (seeexamples). Especially, the monoclonal antibody 9.3 has the best abilityto inhibit tumor growth and invasion of tumor cells of all antibodiestested. Furthermore, the monoclonal antibody 9.3 seems to abolishchemoresistance to a greater extend than the antibody 11A tested in WO2008/046529 (see example 13).

Monoclonal antibodies and the production of monoclonal antibodiesbelongs to the state of the art and is also described in the referencescited in the Materials and Methods section of the examples. In general,monoclonal antibodies can, for example, be prepared in accordance withthe known method of Winter & Milstein (Winter, G. & Milstein, C. (1991)Nature, 349, 293-299). An alternative to preparing monoclonalantibody-secreting hybridomas, a monoclonal antibody directed against apolypeptide of the invention can be identified and isolated by screeninga recombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with the polypeptide of interest. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; WO 90/02809; Fuchs et al., 1991, Bio/Technology9:1370-1372; Hay et al., 1992, Hum. Antibod. Hybridomas 3:81-85; Huse etal., 1989, Science 246:1275-1281; Griffiths et al., 1993, EMBO J.12:725-734.

Since the effect of an antibody is mediated by its capacity to bind aspecific epitope, the invention relates to all monoclonal antibodiesrecognizing the same epitope as the antibody 9.3. Methods fordetermining the epitope of a given antibody are known in the art andinclude the preparation of synthetic linear peptides of a given regionof interest and the subsequent testing whether the antibody binds tosaid peptides (see Epitope Mapping, A practical approach, OxfordUniversity Press 2001, Editors: Olwyn Westwood and Frank Hay).Alternatively, different recombinant proteins covering the region ofinterest can be produced and tested for the binding of the antibody(Oleszewski, M., Gutwein, P., von der Lieth, W., Rauch, U., Altevogt, P.Characterization of the L1-neurocan binding site. Implications for L1-L1homophilic binding. J. Biol. Chem. 275: 34478-34485 (2000).

Furthermore, once a specific epitope of a monoclonal antibody is known,it is within the skill of the person skilled in the art to identify orprepare other antibodies, especially monoclonal antibodies, or bindingmolecules as defined below which bind to the same epitope. For example,it is possible to use the peptides or proteins described above in thecontext of the epitope mapping also for the identification or productionof said antibodies or binding molecules.

As it can be taken from the examples, the epitope of the antibody 9.3 iswith the first immunoglobulin-like domain of L1. Therefore also theepitope of the monoclonal antibody of the invention is preferably withinthe first immunoglobulin-like domain of L1.

In another aspect, the invention relates to an anti-L1 monoclonalantibody, having the same capacity to inhibit tumor growth as themonoclonal antibody 9.3, produced by the hybridoma cell deposited underDSMZ ACC2841. This capacity can be tested by using the same tumor growthassay as described in the Example 1, section 1.3.9. According to theinvention, “the same capacity” means that the monoclonal antibody has atumor growth inhibiting capacity which does not differ more than 5% fromthe tumor growth inhibiting capacity of the monoclonal antibody 9.3.

Preferably, this antibody of the invention also inhibits L1dimerization, as it has been shown for the antibody 5G3 (see above).

In another aspect, the invention relates to an anti-L1 monoclonalantibody, characterized in that at least one of its complementaritydetermining regions (CDRs)

-   -   a) has one of the following sequences RASQDISNYLN, (SEQ ID NO:        1), YTSRLHS, (SEQ ID NO: 2), QQGNTLPWT (SEQ ID NO: 3), RYWML        (SEQ ID NO: 4), EINPRNDRTNYNEKFKT (SEQ ID NO: 5), or GGGYAMDY        (SEQ ID NO: 6) or    -   b) has a sequence which, in comparison to the sequences        mentioned under a) has at least one conservative amino acid        exchange.

The above mentioned sequences show the CDRs of the monoclonal antibody9.3 determined according to the method of Kabat (see Example 2). Such amonoclonal antibody of the invention can, e.g. be produced by CDRgrafting or by recombinant production of the antibody. Such methods areknown in the art (see e.g. Queen, U.S. Pat. No. 5,585,089 and Winter,U.S. Pat. No. 5,225,539, Cabilly U.S. Pat. No. 4,816,567).

In another aspect, the inventions relates to a binding monoclonalcomprising

-   -   a) at least one of the following sequences QDISNY (SEQ ID NO:        7), YTS, QQGNTLPWT (SEQ ID NO: 8), GYTFTRYW (SEQ ID NO: 9),        INPRNDRT (SEQ ID NO: 10), or ALGGGYAMDY (SEQ ID NO: 11) or    -   b) at least one sequence which has in comparison to the        sequences given in a) at least one conservative amino acid        exchange.

These sequences show again the CDRs of the monoclonal antibody 9.3 (seeFIG. 12), but the CDRs have been determined using another method knownin the art, namely according to the IMGT® method from the internationallmMunoGeneTics information Sytem®.

In an especially preferred aspect, the invention relates to a monoclonalantibody, produced by the hybridoma cell deposited under DSMZ ACC2841.This hybridoma cell has been deposited with the Deutsche Sammlung fürMikroorganismen und Zellen on Apr. 25, 2007 under the Budapest Treaty.

In another aspect, the invention relates to a humanized antibody basedon the monoclonal antibody of the invention as described above.

Humanized antibodies are antibody molecules from non-human specieshaving one or more complementarily determining regions (CDRs) from thenon-human species and a framework region (FR) from a humanimmunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089 andWinter, U.S. Pat. No. 5,225,539.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart.

In general, in order to obtain a humanised antibody, nucleic acidsequences encoding human variable heavy chains and variable light chainsmay be altered by replacing one or more CDR sequences of the human(acceptor) sequence by sequence encoding the respective CDR in the mouseantibody sequence (donor sequence). The human acceptor sequence maycomprise FR derived from different genes.

In a preferred embodiment, the humanized antibody of the invention hasat least one non-human CDR and human framework region (FR) residues.

Sequences encoding full length antibodies can be subsequently obtainedby joining the rendered variable heavy and variable light chainsequences to human constant heavy chain and constant light chainregions. Preferred human constant light chain sequences include kappaand lambda constant light chain sequences. Preferred human constantheavy chain sequences include IgG1, IgG2 and sequences encoding IgG1mutants which have rendered immune-stimulating properties. Such mutantsmay have a reduced ability to activate complement and/or antibodydependent cellular cytotoxicity and are described in U.S. Pat. No.5,624,821; WO 99/58572, U.S. Pat. No. 6,737,056. An especially preferredconstant heavy chain is an IgG1 comprising the substitutions E233P,L234V, L235A, A327G, A330S, P331S and a deletion of residue 236.

In another embodiment, the full length antibody comprises an IgA, IgD,IgE, IgM, IgY or IgW sequence.

Suitable human donor sequences can be determined by sequence comparisonof the peptide sequences encoded by the mouse donor sequences to a groupof human sequences, preferably to sequences encoded by human germ lineimmunoglobulin genes or mature antibody genes. A human sequence with ahigh sequence homology, preferably with the highest homology determinedmay serve as the acceptor sequence for the humanization process.

In addition to the exchange of human CDRs for mouse CDRs, furthermanipulations in the human donor sequence may be carried out to obtain asequence encoding a humanized antibody with optimized properties (suchas affinity of the antigen).

In a preferred example, heavy chain residues 31-35, 50-58 and 95-102 andresidues 6, 23, 24, and 49 in the human acceptor sequence are altered tocorrespond to the respective residues of the mouse sequence (Adair, U.S.Pat. No. 5,859,205).

Furthermore the altered human acceptor antibody variable domainsequences may also be rendered to encode one or more amino acids(according to the Kabat numbering system) of position 4, 35, 38, 43, 44,46, 58, 62, 64, 65, 66, 67, 68, 69, 73, 85, 98 of the light variableregion and 2, 4, 36, 39, 43, 45, 69, 70, 74, 75, 76, 78, 92 of the heavyvariable region corresponding to the mouse donor sequence (Carter andPresta, U.S. Pat. No. 6,407,213)

The humanisation of an mouse L1 antibody is described in Example 2.

Also the sequences of the CDRs may be altered, preferably by exchangesleading to a conservative amino acid exchange.

In general, manipulations may result in alterations in the FR as well asthe CDR regions and include exchanges, deletions and insertion ofresidues. The alterations may be induced by random or directedmutagenesis. An antibody phage display system, as described before, maybe employed for the selection of mutants with desired and/or improvedproperties

In another aspect the invention relates to a human antibody capable ofrecognizing the same epitope as the antibody 9.3. Methods for generatinghuman antibodies are known in the art. These methods employ for examplemice in which the endogenous immunoglobuline genes have been partiallyor completely inactivated and human immunoglobuline loci wereintroduced. Upon immunization with an immunogenic epitope, these miceare capable of producing human antibodies (U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,589,369; 5,591,669; 5,625,126; 5,633,425;5,661,016)

In a further preferred embodiment, the humanized antibody of theinvention comprises the sequence of L1_(—)9.3hu or L1_(—)9.3hu3 as shownin FIG. 8 a) and b).

In another aspect, the invention relates to a binding moleculecomprising

-   -   a) at least one of the following sequences RASQDISNYLN (SEQ ID        NO: 1), YTSRLHS (SEQ ID NO: 2), QQGNTLPWT (SEQ ID NO: 3), RYWML        (SEQ ID NO: 4), EINPRNDRTNYNEKFKT (SEQ ID NO: 5), or GGGYAMDY        (SEQ ID NO: 6) or    -   b) at least one sequence which has in comparison to the        sequences given in a) at least one conservative amino acid        exchange.

As explained above, these sequences show the CDRs of the antibody 9.3(see Example 2).

In another aspect, the invention relates to a binding moleculecomprising

-   -   a) at least one of the following sequences QDISNY (SEQ ID NO:        7), YTS, QQGNTLPWT (SEQ ID NO: 8), GYTFTRYW (SEQ ID NO: 9),        INPRNDRT (SEQ ID NO: 10), or ALGGGYAMDY (SEQ ID NO: 11) or    -   b) at least one sequence which has in comparison to the        sequences given in a) at least one conservative amino acid        exchange.

As explained above, these sequences show again the CDRs of themonoclonal antibody 9.3, determined by another method known in the art.

According to the invention, a binding molecule is a molecule capable ofbinding L1. Preferably, the binding molecule is an immunoglobulincomprising molecule, i.e. comprises at least one Ig domain.

In another aspect, the inventions, the binding molecule of the inventionis selected from the group consisting of single chain antibodies (e.g.scFv, multimers of scFv like diabodies, triabodies or tetrabodies,antibody fragments (e.g. Fab), tandabs, flexibodies, bispecificantibodies, and chimeric antibodies.

The structure of an antibody and especially the function of its CDRs isknown in the art (Carter P J. Potent antibody therapeutics by design.Nature Rev. Immunol. 6:343-357, 2006).

scFv and multimers thereof, tandabs, diabodies and flexibodies arestandard antibody formats known in the art, e.g. from WO 88/1649, WO93/11161, WO 99/57150 and EP1293514B1.

In single chain Fv (scFv) the two antigen binding variable regions ofthe light and heavy chain (VH Fv and VL Fv) of an antibody areartificially connected by a linker peptide, designated as single chainvariable fragment or single chain antibody (Bird, et al. (1988) Science242:423-426; Orlandi, et al (1989) Proc Natl Acad Sci USA 86:3833-3837;Clarkson et al., Nature 352: 624-628 (1991)). The antigen binding siteis made up of the variable domains of light and heavy chains of amonoclonal antibody. Several investigations have shown that the Fvfragment has indeed the full intrinsic antigen binding affinity of onebinding site of the whole antibody.

In the context of this invention, diabodies are scFv with two bindingspecificities and can either be monospecific and bivalent or bispecificand bivalent.

Tandabs and flexibodies are further antibody formats which are e.g.defined in US2007031436 and EP1293514, respectively.

Antibody fragments that contain the idiotypes of the protein can begenerated by techniques known in the art. For example, such fragmentsinclude, but are not limited to, the F(ab′)2 fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′ fragmentthat can be generated by reducing the disulfide bridges of the F(ab′)2fragment; the Fab fragment that can be generated by treating theantibody molecular with papain and a reducing agent; and Fv fragments.

A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss etal., U.S. Pat. No. 4,816,397).

Bifunctional, or bispecific, antibodies have antigen binding sites ofdifferent specificities. Various forms of bispecific antibodies havebeen produced. These include BSIgG, which are IgG molecules comprisingtwo distinct heavy chains and two distinct light chains that aresecreted by so-called “hybrid hybridomas”, and heteroantibody conjugatesproduced by the chemical conjugation of antibodies or antibody fragmentsof different specificities (Segal D M, Weiner G J, Weiner L M.

Bispecific antibodies in cancer therapy. Current Opin. Immunol.11:558-562, 1999, Van Spriel A B, Van Ojik H H, Van de Winkel J G J.Immunotherapeutic perspective for bispecific antibodies. ImmunologyToday 21:391-397, 2000).

Bispecific antibodies have been generated to deliver cells, cytotoxins,or drugs to specific sites. An important use has been to deliver hostcytotoxic cells, such as natural killer or cytotoxic T cells, tospecific cellular targets. (P. J. Lachmann, Clin. Exp. Immunol. 79: 315(1990)). Another important use has been to deliver cytotoxic proteins tospecific cellular targets. (V. Raso, T. Griffin, Cancer Res. 41:2073(1981); S. Honda, Y. Ichimori, S. Iwasa, Cytotechnology 4:59 (1990)).Another important use has been to deliver anti-cancer non-protein drugsto specific cellular targets (J. Corvalan, W. Smith, V. Gore, Intl. J.Cancer Suppl. 2:22 (1988); M. Pimm et al., British J. of Cancer 61:508(1990)). Such bispecific antibodies have been prepared by chemicalcross-linking (M. Brennan et al., Science 229:81 (1985)), disulfideexchange, or the production of hybrid-hybridomas (quadromas). Quadromasare constructed by fusing hybridomas that secrete two different types ofantibodies against two different antigens (Kurokawa, T. et al.,Biotechnology 7.1163 (1989)).

In a preferred embodiment of the invention, the antibody or bindingmolecule of the invention is linked to an active substance, preferably atoxin, a nanoparticle, a cytokine, or a radionuclide. Such antibodyconjugates are known in the art (Wu A M, Senter P D. Arming antibodies:prospects and challenges for immunoconjugates. Nature Biotechnol.23:1137-1146, 2005, Pastan I, Hassan R, FitzGerald D J, Kreitman R J.Immunotoxin treatment of cancer. Annu. Rev. Med. 58:221-237, 2007, WO90/12592, WO 2007/030642, WO 2004/067038, WO 2004/003183, US2005/0074426, WO 94/04189).

In a preferred embodiment, the antibody or binding molecule of theinvention binds L1 with an affinity (KD) of at least 10⁻⁸, preferably ofat least 10⁻⁹, more preferably of at least 10⁻¹⁰ or 10⁻¹¹.

Preferably, the antibody of the invention does not significantly bind toother members of the L1-protein family as for example CHL1 (closehomolog of L1, accession number NM_(—)006614), NrCAM (Neuronal celladhesion protein, accession number NM_(—)001037132 or NM_(—)005010)and/or NFASC (Neurofascin, accession number NM_(—)015090). Preferablythe antibody binds the other members of the L1-family with an at least100-fold lower affinity, more preferably at least 1000-fold loweraffinity compared to the affinity for L1. The affinity of the antibodyfor the different proteins can be determined for example by measuringthe binding affinity to recombinant proteins as described in example 6.The binding of the antibody to the different L1 family members of theL1-family may also be determined by expressing said proteins on CHOcells and measuring the antibody binding by FACS analysis as describedin Example 1.2 and Example 7.

It is one aspect of the invention that the antibody does notsignificantly increase the release of cytokines, e.g. tumour necrosisfactor-alpha or interferon gamma. Preferably the release is notincreased by more than 30%, more preferably not more than 20% and mostpreferably not more than 10%. The release of cytokines can be tested asdescribed in Example 8. Alternatively the concentration of cytokines canbe determined in the blood of an animal before and after theadministration of the antibody. The cytokine concentration may bedetermined by an ELISA assay or other methods known in the art.

In another preferred embodiment the antibody does not significantlyinduce T-cell proliferation or inhibit T-cell proliferation. The effectof an antibody on T-cell proliferation can be determined as described inExample 9.

The invention further relates to a binding molecule which is capable ofbinding to the same L1 epitope recognized by the monoclonal antibody9.3, produced by the hybridoma cell deposited under DSMZ ACC2841.Preferably, with respect to this binding molecule of the invention, thesame embodiments defined with respect to the structure of the bindingmolecule described above also apply to this binding molecule of theinvention.

Preferably, the binding of the antibody to the epitope is notsignificantly increased or decreased by the glycosylation state of theL1 protein. The influence of the glycosylation state on the antibodybinding can be determined as described in Example 10.

Furthermore, the invention relates to a hybridoma cell that produces themonoclonal antibody of the invention.

Furthermore, the invention relates to the hybridoma cell deposited underDSMZ ACC2841.

As explained above and as described in the example section, themonoclonal antibody or binding molecule of the invention is especiallysuitable for the treatment of tumorigenic diseases.

Therefore, in another aspect, the invention relates to the use of theantibody of the invention or the binding molecule of the invention forthe preparation of a medicament for the treatment of a tumorigenicdisease.

Furthermore, the invention also relates to a method for treating atumorigenic disease, wherein an antibody or binding molecule of theinvention is administered to a subject in an effective amount to treatsaid disease. With respect to said method of the invention, allembodiments as defined below for the use of the invention also apply.

As mentioned above, in the art it has been suggested to use anti-L1antibodies for sensitizing tumor cells for the treatment with achemotherapeutic drug or with radiotherapy (see Sebens Müerkoster etal., Oncogene. 2007 Apr. 26; 26(19):2759-68, Epub 2006 Nov. 6).Consequently, in another aspect, the present invention relates to theuse of the antibody of the invention or the binding molecule of theinvention for sensitizing tumor cells in a patient for the treatmentwith a chemotherapeutic drug or with radiotherapy.

This aspect of the present invention is especially useful in cases wherethe tumor cells are at least partially resistant to chemotherapy or toradiotherapy.

Therefore, in a preferred embodiment of the invention, the cells to besensitized are at least partially resistant to the treatment with saidchemotherapeutic drug or to radiotherapy.

In the context of the present invention, the term “sensitizing” is to beunderstood that after the treatment with the anti L1 antibody or bindingmolecule of the invention, the tumor cells are more susceptible to thetreatment with a chemotherapeutic drug or with radiotherapy than beforesaid treatment. This can e.g. be tested by isolating tumor cells fromthe patient and testing in vitro whether the treatment with saidantibody or binding molecule of the invention results in a sensitizationof the cells. This test can be performed as described in reference(Sebens Müerkoster et al., Oncogene. 2007 Apr. 26; 26(19):2759-68, Epub2006 Nov. 6).

In a preferred embodiment, the cells, before the administration of theanti L1 antibody or binding molecule of the invention, were notsusceptible to the treatment or only susceptible to an extend that thetreatment with a chemotherapeutic drug or with radiotherapy would notresult in the desired therapeutic effect.

Preferably, with the help of the anti-L1 antibody or binding molecule ofthe invention, the susceptibility is increased by at least 20%, morepreferably by at least 40% and even more preferably by at least 100%.

An overview over chemotherapeutic drugs and radiotherapy is e.g. givenin Remmington's Pharmaceutical Sciences, 5^(th) ed., chapter 33, inparticular pages 624 to 652.

Any of numerous chemotherapeutic drugs can be used in the methods oruses of the invention. These compounds fall into several differentcategories, including, for example, alkylating agents, antineoplasticantibiotics, antimetabolites, and natural source derivatives.

Examples of alkylating agents that can be used in the invention includebusulfan, caroplatin, carmustine, chlorambucil, cisplatin,cyclophosphamide (i.e., cytoxan), dacarbazine, ifosfamide, lomustine,mecholarethamine, melphalan, procarbazine, streptozocin, and thiotepa.

Examples of antineoplastic antibiotics include bleomycin, dactinomycin,daunorubicina, doxorubicin, idarubicin, mitomycin (e.g., mitomycin C),mitoxantrone, pentostatin, and plicamycin.

Examples of antimetabolites include fluorodeoxyuridine, cladribine,cytarabine, floxuridine, fludarabine, fluorouracil (e.g., 5-fluorouracil(5FU)), gemcitabine, hydroxyurea, mercaptopurine, methotrexate, andthioguanine.

Examples of natural source derivatives include docetaxel, etoposide,irinotecan, taxanes (e.g. paclitaxel), teniposide, topotecan,vinblastine, vincristine, vinorelbine, prednisone, and tamoxifen.

Additional examples of chemotherapeutic agents that can be used in theinvention include asparaginase and mitotane.

Furthermore, also C2 ceramide can be used.

In an especially preferred embodiment, the chemotherapeutic drug isselected from the group consisting of actinomycin-D, mitomycin C,cisplatin, doxorubicin, etoposide, verapamil, podophyllotoxin, 5-FU,taxans such as paclitaxel, and carboplatin.

According to the invention, the term “radiotherapy” refers to eachradiation therapy which is commonly used to treat tumors cells. In apreferred embodiment, this therapy include γ-rays, X-rays, microwaves,UV radiation as well as the direct delivery of radio-isotopes to or nextto tumor cells (brachytherapy).

As mentioned above, the object of this aspect of the invention is tosensitize tumor cells for the treatment with a chemotherapeutic drug orwith radiotherapy. Consequently, in a preferred embodiment, after thesensitization with the anti L1 antibody or binding molecule of theinvention, the patient is further treated with said chemotherapeuticdrug or with said radiotherapy.

In the context of the present invention, it is envisaged to sensitizetumor cells of any cell type or to treat any tumorigenic disease.Preferably, the tumor cells or the tumorigenic disease are of a typeselected from the group consisting of astrocytoma, oligodendroglioma,meningioma, neurofibroma, glioblastoma, ependymoma, Schwannoma,neurofibrosarcoma, medulloblastoma, melanoma, pancreatic cancer,prostate carcinoma, head and neck cancer, breast cancer, lung cancer,ovarian cancer, endometrial cancer, renal cancer, neuroblastomas,squamous cell carcinomas, medulloblastomas, hepatoma, colon cancer, andmesothelioma and epidermoid carcinoma.

Furthermore, it is preferred that the tumor cells are from an epithelialtumor or the tumorigenic disease is an epithelial tumor, preferablywherein the epithelial tumor is pancreatic cancer, colon cancer, ovariancancer or endometrial cancer.

In a preferred embodiment the antibody does not induce neuronal sideeffects when administered in a therapeutically effective amount.

As discussed above, the anti-L1 antibody or binding molecule are usedfor the preparation of a pharmaceutical composition.

In general, the pharmaceutical compositions of the present inventioncomprise a therapeutically effective amount of a therapeutic, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly, in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, including but not limited to peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred carrier whenthe pharmaceutical composition is administered orally. Saline andaqueous dextrose are preferred carriers when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions are preferably employed as liquidcarriers for injectable solutions. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsions,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thetherapeutic, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated, in accordancewith routine procedures, as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water or saline forinjection can be provided so that the ingredients may be mixed prior toadministration.

The therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freecarboxyl groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., those formed with free aminegroups such as those derived from isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc., and those derived fromsodium, potassium, ammonium, calcium, and ferric hydroxides, etc.

The amount of the therapeutic of the invention, which will be effectivein the treatment of a particular disorder or condition, will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.In general, suppositories may contain active ingredient in the range of0.5% to 10% by weight; oral formulations preferably contain 10% to 95%active ingredient.

Various delivery systems are known and can be used to administer atherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, and microcapsules: use of recombinant cells capable ofexpressing the therapeutic, use of receptor-mediated endocytosis (e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432); construction of atherapeutic nucleic acid as part of a retroviral or other vector, etc.Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion, by bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oral,rectal and intestinal mucosa, etc.), and may be administered togetherwith other biologically active agents. Administration can be systemic orlocal. In addition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysnitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment. This may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

In another embodiment, the therapeutic can be delivered in a vesicle, inparticular a liposome (Langer, 1990, Science 249:1527-1533), moreparticular a cationic liposome (WO 98/40052).

In yet another embodiment, the therapeutic can be delivered via acontrolled release system. In one embodiment, a pump may be used(Langer, supra). In yet another embodiment, a controlled release systemcan be placed in proximity of the therapeutic target, thus requiringonly a fraction of the systemic dose.

Within the context of this aspect of the invention, the invention alsoincludes a method for sensitizing tumor cells in a patient for thetreatment with a chemotherapeutic drug or with radiotherapy, comprisingadministering to the patient an efficient amount of an anti-L1 antibodyor binding molecule of the invention. All embodiments described abovealso apply to this method of the invention.

Throughout the invention, the term “effective amount” means that a givenmolecule or compound is administered in an amount sufficient to obtain adesired therapeutic effect. In case that, throughout the invention, twocompounds are administered in a therapeutic effective amount, thisincludes that one or each of the compounds is administered in asubtherapeutic amount, i.e. that the amount of each compound on its ownis not sufficient to provide a therapeutic effect, but that thecombination of the compounds results in the desired therapeutic effect.However, it is also included within the present invention that each ofthe compounds on its own is administered in a therapeutically effectiveamount.

In another aspect of the invention, the invention relates to the use ofthe anti-L1 antibody or binding molecule of the invention for thepreparation of a medicament for the treatment of tumor cells in apatient previously treated with a chemotherapeutic drug or withradiotherapy.

As mentioned above, the treatment of tumor cells with anti-L1 antibodieshas already been described in WO 02/04952 and WO 06/013051, incorporatedherein by reference.

In the context of the present invention, the term “previously treated”may include patients which have already been treated with achemotherapeutic drug or with radiotherapy in the course of a separatedregimen which has taken place e.g. within the last six or eight months.

In the course of tumor treatment with chemotherapeutic drugs orradiotherapy it is in most cases observed that after an initial responseof the tumor to such therapy (tumor mass reduction or stabilization ofthe disease) the tumors start to progress again. Such progressionusually starts upon weeks or months after such therapy. Typically thesetumors are then resistant to further treatment with the previouslyapplied chemotherapeutic drug and other treatment modalities are wanted.As described above it has been found that such resistant tumors expressL1 and therefore become a target for anti-L1 antibodies.

Therefore, according to this embodiment of the invention, the term“previously treated” preferably means that the patient previouslyreceived such treatment, such treatment showed an initial effect and—atthe time of therapy with the anti-L11 antibody or the binding moleculethe tumor is progressing again.

Furthermore, the term “previously treated” may also be seen in a contextwhere the L1 anti-L1 antibody or the binding molecule and thechemotherapeutic drug or radiotherapy are used within the same regimen,meaning that the treatments are given within one treatment schedule. Inthis context “in one treatment schedule” means that the treatment areapplied at the same time, one after another or intermittently, but—incontrast to above—time distances between the individual treatments areshort (within one week or within 2-4 days) and, if a treatment successis seen, one does not wait for tumor progression before the nexttreatment is applied.

Preferably, in this context, the invention includes the case where apatient is treated with a chemotherapeutic drug or with radiotherapy andsubsequently, preferably within one week or less and more preferablywithin 2-4 days, a treatment with the anti-L1 antibody or the bindingmolecule of the invention is started. In a further preferred embodimentseveral cycles of chemotherapy or radiotherapy on one side and treatmentwith the anti-L1 antibody or the binding molecule are made, withintervals of preferably one week or less and more preferably within 2-4days.

In a preferred embodiment, the patient is at least partially resistantto the treatment with said chemotherapeutic drug or with radiotherapy,an effect often observed in the course of said treatment types (seeabove).

In a further aspect, the invention relates to the use of the anti-L1antibody or the binding molecule of the invention for the preparation ofa medicament for the treatment of tumor cells in a patient at leastpartially resistant to treatment with a given chemotherapeutic drug orwith radiotherapy.

In the context of the present invention, the term “resistant totreatment” means that the respective tumor cell does not react to thetreatment with a chemotherapeutic drug or with radiotherapy in acomplete manner. Rather, with respect to this tumor cell, treatment withsaid chemotherapeutic drug or radiotherapy is rather ineffective or evenshows no effects.

In a further aspect of the invention, the invention relates to the useof the anti-L1 antibody or the binding molecule of the invention for thepreparation of a medicament for the treatment of a tumorigenic disease,wherein the anti-L11 antibody or the binding molecule is administered incombination with a chemotherapeutic drug or with radiotherapy,preferably wherein the chemotherapeutic drug or the radiotherapy isadministered prior to the anti-L11 antibody or binding molecule of theinvention.

According to the invention, the term “treatment of tumorigenic disease”includes both the killing of tumor cells, the reduction of theproliferation of tumor cells (e.g. by at least 30%, at least 50% or atleast 90%) as well as the complete inhibition of the proliferation oftumor cells. Furthermore, this term includes the prevention of atumorigenic disease, e.g. by killing of cells that may or a prone tobecome a tumor cell in the future as well as the formation ofmetastases.

According to the invention, the term “in combination with” includes anycombined administration of the anti-L1 antibody or the binding moleculeand the chemotherapeutic drug of radiotherapy. This may include thesimultaneous application of the drugs or radiotherapy or, preferably, aseparate administration. In case that a separate administration isenvisaged, one would preferably ensure that a significant period of timewould not expire between the time of delivery, such that the anti-L1antibody or the binding molecule and the chemotherapeutic drug orradiotherapy would still be able to exert an advantageously combinedeffect on the cell. In such instances, it is preferred that one wouldcontact the cell with both agents within about one week, preferablywithin about 4 days, more preferably within about 12-36 hours of eachother.

The rational behind this aspect of the invention is that theadministration of chemo-therapeutic drugs or the treatment withradiotherapy leads to an increase of L1 expression on the surface of thetumor cells which in turn makes the tumor cells a better target for theanti-L1 antibody or the binding molecule.

Therefore, this aspect of the invention also encompasses treatmentregimens where an the anti-L1 antibody or the binding molecule isadministered in combination with the chemotherapeutic drug orradiotherapy in various treatment cycles wherein each cycle may beseparated by a period of time without treatment which may last e.g. fortwo weeks and wherein each cycle may involve the repeated administrationof the anti-L11 antibody or the binding molecule and/or thechemotherapeutic drug or radiotherapy. For example such treatment cyclemay encompass the treatment with a chemotherapeutic drug or withradiotherapy, followed by e.g. the twice application of the anti-L1antibody or the binding molecule within 2 days.

Throughout the invention, the skilled person will understand that theindividual therapy to be applied will depend on the e.g. physicalconditions of the patient or on the severity of the disease and willtherefore have to be adjusted on a case to case basis.

Especially in the course of such repeated treatment cycles, it is alsoenvisaged within the present invention that the anti-L1 antibody or thebinding molecule is administered prior to the chemotherapeutic drug orthe radiotherapy.

In the context of the above aspects of the invention, the invention alsorelates to a method for treating tumor cells in a patient previouslytreated with a chemotherapeutic drug or with radiotherapy, comprisingadministering to the patient a therapeutically effective amount of theanti-L1 antibody or binding molecule of the invention. Furthermore, theinvention relates to a method for treating tumor cells in a patient atleast partially resistant to treatment with a given chemotherapeuticdrug or with radiotherapy, comprising administering to the patient atherapeutically effective amount of the anti-L1 antibody or bindingmolecule of the invention. Furthermore, the invention relates to amethod for treating tumor cells in a patient, comprising administeringto the patient a therapeutically effective amount of the anti-L1antibody or binding molecule of the invention in combination with achemotherapeutic drug or with radiotherapy. Furthermore, Furthermore,the invention relates to a method for treating tumor cells in a patient,comprising administering to the patient a therapeutically effectiveamount of the anti-L1 antibody or binding molecule of the invention.

The antibody of the invention may also be used in a method for adiagnostic method to determine the level of the L1 protein in bodytissues or fluids.

With respect to these methods of the invention, all embodimentsdescribed above for the other uses or methods of the invention alsoapply.

The invention also relates to the antibody or the binding molecule ofthe invention for use as a medicament for the treatment of a tumorigenicdisease or for sensitizing of tumor cells in a patient for the treatmentwith a chemotherapeutic drug or with radiotherapy.

In a preferred embodiment, said use exhibits further the features asdefined for the uses of the invention.

The invention also relates to pharmaceutical compositions comprising theantibody or binding molecule of the invention. With respect to saidpharmaceutical composition, all embodiments described above also apply.

The invention is further illustrated by the following figures andexamples which are not intended to limit the scope of the invention.

LEGENDS TO FIGURES AND TABLES

FIG. 1

(A) FACS analysis of CHO, CHO-L1, SKOV3ip and OVMz cells. Cells werestained with the indicated mAbs (10 μg/ml) for 30 min at 4° C. Followedby a secondary PE-conjugated mAb. (B) Western blot analysis. Celllysates from CHO wt, CHO-L1, OVMz and SKOV3ip cells were transferred ona PVDF membrane and then incubated with the indicated mAb to L1 (1μg/ml), followed by a POX-conjugated secondary mAb.

FIG. 2

(A) Effect of antibodies on Erk-phosphorylation in SKOV3ip cells. Cellswere incubated for 24 h at 37° C. with the indicated purified antibodiesto L1 (10 μg/ml) or isotype control IgG1. Cells were also treated withDMSO (vehicle), or the MEK-specific inhibitor PD59098. Cell lysates wereexamined for phosphorylation of Erk.

(B) Effect of antibodies on Erk phosphorylation in SKOV3ip cells.Fluorescent staining of antibody treated cells with a phospho-Erkspecific antibody and an Alexa488-conjugated secondary mAb.

FIG. 3

Analysis of matrigel cell invasion. Antibody (10 μg/ml) treated SKOV3ipcells were seeded into a 4-well plate and allowed to invade into thematrigel for 20 h (5% CO₂; 37° C.).

FIG. 4

Differential gene expression in SKOV3ip cells. (A) SKOV3ip cells weretransfected with L1-specific or scrambled siRNA and 72 h later mRNAswere isolated, transcribed to cDNA and used as template for qPCR(SYBRgreen analysis). (B) SKOV3ip cells were treated with the L1-9.3 mAb(10 μg/ml) or the control mAB IgG1 (10 μg/ml) and 96 h later mRNAs wereisolated, transcribed to cDNA and analyzed by qPCR for the expression ofthe indicated genes (SYBRgreen analysis). (C) Differential geneexpression of residual tumor cells. mRNAs from residual tumors wereisolated from antibody treated animals, transcribed into cDNA andanalyzed by qPCR for the expression of the indicated genes.

FIG. 5

Tumor growth in nude mice. LacZ-tagged SKOV3ip cells were injected i.p.into nude mice and after tumor implantation animals were treated withthe indicated L1 mAbs or control mAb EpCAM (Hea125). After 30 days thetumor volume was determined and is given as the ratio between X-Galstained tumormass and the total situs. 6 animals were analyzed pergroup.

FIG. 6

(A) Western blot analysis of L1-V5 constructs. Supernatant oftransfected Sf9 insect cells were received from Ricardo Gouveia andanalyzed by Western blot using L1-9.3 mAb and reprobed by anti-V5 mAb.(B) Western blot analysis of L1-FC constructs. L1-FC constructs weretransfected into Cos-7 cells using Jet PEI™ transfection reagent asdescribed. After 3 days supernatants were purified using SepharoseA andanalyzed by Western blot using L1-9.3 mAb.

FIG. 7

Homophilic cell adhesion assay. (A) The binding of J558-L1 cells wasanalyzed by bright field microscopy. One example of each treatment isshown here. In the red box coating with L1-Fc (10 μg/ml) is highlightedand in the black box the both controls, fibronectin (10 μg/ml) and BSA,are shown. (B) The graph shows the mean±SD of bound cells after theindicated antibody or control treatment.

FIG. 8

The antibody light chain and heavy chain DNA sequences used to constructthe humanized antibodies are provided in FIGS. 8 a (SEQ ID NOS 17-21, inrespectively, in order of appearance) and 8 b (SEQ ID NOS 22-25,respectively, in order of appearance) respectively.

FIG. 9

Amino acid sequences of: (a) the murine L1_(—)9.3 scFv (SEQ ID NO: 26);(b) the humanized L1_(—)9.3Hu (SEQ ID NO: 27); and (c) L1_(—)9.3Hu3scFvs (SEQ ID NO: 28).

FIG. 10

DNA and amino acid sequences of the expressed portions of constructs:(a) L1_(—)9.3 (SEQ ID NOS 29-30, respectively); (b) L1-9.3Hu (SEQ ID NOS31-32, respectively); and (c) L1_(—)9.3Hu3 scFv (SEQ ID NOS 33-34,respectively ).

FIG. 11

Binding of the L1_(—)9.3, L1-9.3Hu and L1_(—)9.3Hu3 scFvs to the humanL1 cancer antigen. Rows A, B and C are coated with L1 and rows D, E andF are coated with streptavidin. The blue colour in the wells indicatesbinding of the individual scFv to the L1 on the plate. The lack ofcolour in the streptavidin coated rows shows that the single chainantibodies are specifically binding to L1

FIG. 12

Genomic sequences of the variable domains of the monoclonal antibody 9.3

a) Sequence of the kappa chain variable region (SEQ ID NO: 35) (dottedlines: CDR1, dashed lines: CDR2, underlined: CDR3)

b) Sequence of the heavy chain variable region (SEQ ID NO: 36) (dottedlines: CDR1, dashed lines: CDR2, underlined: CDR3)

FIG. 13

A) Human PBMC and L1-positive OVMZ tumor cells were incubated withL1-9.3 mAb for 24 h and the amount of bound antibody was determined byFACS analysis.

B) The dissociation constants K_(D) were estimated from the regressioncurves using the concentration at half-maximal binding.

FIG. 14

L1-9.3 has no effect on the release of cytokines by resting andactivated human PBMC. Cytokine levels of resting and OKT3-activated PBMCfrom three different donors were determined after an incubation for 24 hin presence or absence of 20 μg/ml L1-9.3. Ionomycin/PMA and LPS wereused as stimulation controls. Results for IFN-γ (A) and TNF-α (B) areshown.

FIG. 15

L1-9.3 does not induce T cell proliferation and has no effect onOKT3-induced T cell proliferation. Proliferation of OKT3-activated PBMCfrom two different donors was determined in presence or absence of 20μg/nm L1-9.3 using a BrdU incorporation assay 48 h post stimulation.There was no difference, whether the antibody was added prior, inparallel or after stimulation with 75 ng/ml OKT3. L1-9.3 by itself didnot result in T cell activation.

FIG. 16

L1-9-3 was unaffected by deglycosylation of L1. The Western blotstaining of L1 in untreated and deglycosylated cell lysate is shownusing several different anti-L1 mAbs. The tested antibodies can bedivided into three classes in respect to their glycosylation-dependency:First class (unaffected by glycosylation): L1-9.3. Second class (bindingin WB was negatively affected by deglycosylation): 11A, 14.10, OV52.24and OV549.20. Third class (binding in WB was positively affected bydeglycosylation): 35.9 and 38.12.

FIG. 17

The figure shows in vivo binding of intravenously applied L1-9.3 tocollecting ducts of the kidney. In vivo binding was only detectableusing the amplification system CSA (FIG. 17A), while by using theconventional ABC-method, no signal was visible (FIG. 17B). Hence, L1-9.3was detected in a range of 30-300 pmol in the tissue (L1-9.3concentration is presumably higher than 5 ng/ml and below 50 ng/ml).Negative control did not show staining, thus, unspecific staining can beexcluded (FIG. 17C). The staining pattern of in vivo bound L1-9.3 (FIG.17A) corresponds to the L1 expression pattern in the kidney whendirectly staining tissue sections with L1-9.3 (FIG. 171D).

FIG. 18

FACS Analysis of Humanized L1-9.3 mAbs

Flow cytometry analysis of SKOV3ip pcDNA3.1 luciferase cells. Cells werestained with the indicated humanized mAbs (10 μg/ml) for 30 min 4° C.,followed by a secondary PE-conjugated mAb.

FIG. 19

Mouse SKOV3ip Xenograft-Model

7*10⁶ SKOV3ip pcDNA3.1 luciferase cells were injected intraperitonealinto 6 weeks old CD1 nu/nu female mice. After 24 h mice were randomizedin groups of 10 mice. Each group of mice was three times weekly injectedwith 300 μg either mAb L1-chi9.3, mAbL1-hu3 or PBS intraperitoneally.

On day 33 mice were imaged (FIG. 2). Tumor volume was determined usingthe XENOGEN IVIS 200 System. In brief, mice were anesthetised andinjected with 100 μl Luciferin D (3 kg/mouse) intraperitoneally.Afterwards, luciferase activity of the tumor cells was measured bydetecting light emission. The tumor volume is shown as photon per second(total flux). Statistical analysis was done using the student's t-test.

FIG. 20

In Vivo Total Tumor Mass

After 36 days mice were sacrificed and the tumor mass was determined.Tumor growth is given as a ratio of tumor mass to bodyweight. (Aindividual mice, B mean value). Statistical analysis was done using thestudent's t-test. Thus, the treatment of immundeficient mice with L19.3antibody could be reproduced with chimarised and humanized forms of theL1 9.3 mAb.

FIG. 21

PT45-P1res cells were either left untreated (w/o) or were treated with20 μg/mL gemcitabone (A) or etoposide (B) in the absence (w/o) orpresence of either 1 or 10 μg/mL anti L1CAM antibody 9.3 or 1 or 10μg/mL isotype matched control antibody. After 24 hours, cells wereanalysed by caspase-3/-7 assay. Means±SD from three independentexperiments are shown. * indicates p<0.05.

FIG. 22

Colo357 cells were either left untreated (w/o) or were treated with 20μg/mL gemcitabone (A) or etoposide (B) in the absence (w/o) or presenceof either 1 or 10 μg/mL anti L1CAM antibody 9.3 or 1 or 10 μg/mL isotypematched control antibody. After 24 hours, cells were analysed bycaspase-3/-7 assay. Means±SD from three independent experiments areshown. * indicates p<0.05.

Table 1

The table shows a summary of antibodies tested in the indicated assays.

EXAMPLES 1. Example 1

1.1 Summary of Example 1

The L1 adhesion molecule (L1-CAM) is a transmembrane cell adhesionmolecule involved in cell migration and axon guidance in the developingnervous system. L1 is also over-expressed in ovarian and endometrialcarcinomas. Here L1 expression is associated with poor prognosis. Incarcinoma cell lines, L1 over-expression augments cell motility, tumorgrowth in mice and induces expression of Erk-dependent genes. Here weshow that treatment with antibodies to L1 abrogates Erk-activation,blocks cell invasion to matrigel and decreases tumor growth in nudemice. In cells treated with L1 antibodies the induction of Erk-dependentgenes such as HOX A9, β3 integrin and IER 3 are reversed in vitro and invivo. In this report, we demonstrate that the antibody L1-9.3 is thebest therapeutic antibody of all tested L1 antibodies. In all casesL1-9.3 showed the best results concerning the invasive phenotype ortherapeutic effect on tumor growth. We could show that L1-9.3 binds tothe first Ig-like domain of L1 and can block the L1-L homophilicbinding. The blocking of homophilic binding was only observed withL1-9.3. We conclude, that L1-9.3 is superior in therapy as it combinestwo functions: it blocks erk activation and interferes with the bindingfunction of L1.

1.2 Results of Example 1

1.2.1 FACS Analysis of the new L1 Antibodies

Using immunization with a recombinant L1-Fc fusion protein, we generatednovel L1 antibodies L1-9.3, L1-14.10, L1-35.9 and L1-38.12. To elucidatethe specificity for L1 the new L1 mAbs were tested these antibodies onthe endogenous L1 expressing ovarian carcinoma cell lines OVMz andSKOV3ip and the chinese hamster ovary cells CHO and stably transducedCHO-L1 cells by fluorescent staining (FIG. 1A) and Western blot analysis(FIG. 1B). All tested antibodies showed a positive staining of L1 inCHO-L cells (FIG. 1A). The staining pattern for the OVMz and the SKOV3ipcells was different for the antibodies. Interestingly, the L1-9.3antibody showed bright staining of both ovarian carcinoma cell linesOVMz and SKOV3ip, whereas the L1-14.10 showed a very weak staining (FIG.1A). The two L1 antibodies L1-35.9 and L1-38.12 could not bind to theendogenous L1 of these cells (FIG. 1A). As expected, no staining for L1could be observed in CHO cells which we used as negative control. Allnew antibodies detected the full length L1 in CHO-L1, OVMz and SKOV3ipcell lysates by Western blot analysis. The L1-negative CHO cells servedagain as negative control.

1.2.2 The Erk Phosphorylation is Decreased after Antibody Treatment

A recent report has shown that expression of L1 in cooperation withserum-derived growth factors lead to sustained Erk-activation and theinduction of Erk-dependent genes (Silletti et al, 2004). We investigatedif the suppressive effect of L1-antibodies might be due to interferencewith L1-mediated gene regulation. Therefore we examined the mode ofaction of L1 antibodies using SKOV3ip cells. The mAbs L1-11A, L1-9.3 andL1-14.10 efficiently blocked Erk-phosphorylation (FIG. 2A) in vitro.There was no inhibition with isotype matched control mAb, DMSO asvehicle or the L1 antibody L1-38.12 (FIG. 2A) that can bind only theneural isoform of L1. Fluorescent analysis with the phospho-specific Erkantibody confirmed a clear reduction of activated Erk. A depletion fromthe nucleus in L1-mAb treated cells (L1-11A, L1-9.3 and L1-14.10) couldalso be observed (FIG. 2B).

1.2.3 Antibody Treatment with L1-Antibodies Reduced Cell Invasion

It has been demonstrated before that treatment with an antibody to L1(L1-11A) reduced the haptotactic cell migration on fibronectin and thematrigel invasion of different cell lines (Arlt et al, 2006). Wecompared the invasion capacity of SKOV3ip cells treated with thedifferent L1 antibodies. The antibodies L1-11A, L1-14.10 and especiallyL1-9.3 reduced the invasion of the SKOV3ip (FIG. 3). In sharp contrast,cells treated with the antibodies L1-35.9 or L1-38.12 did not show areduction of invasion (FIG. 3).

1.2.4 Antibodies to L1 Affect Gene Expression In Vitro and In Vivo

We further examined whether antibodies to L1 affect the gene expressionprofile in SKOV3ip cells in vitro in a similar fashion as observed forsiRNA-mediated depletion of L1 (FIG. 4A). Indeed, qRT-PCR analysis ofcells treated with L1-9.3 or L1-11A versus control antibody showedsignificant changes in the expression of L1-regulated genes such as β3integrin, the transcription factors HOXA9 and the apoptosis-relatedgenes IER 3 and STK 39 (FIG. 4A). The same set of genes wasdownregulated in SKOV3ip cells transduced with a L1-specific siRNA (FIG.4B).

We tested whether mAb L1-9.3 could also influence the gene expressionprofile of SKOV3ip cells in vivo similar to that observed in vitro. Tothis end, mRNA from residual tumors of L1-9.3 treated mice or IgGcontrol treated mice were isolated and subjected to qRT-PCR analysis.L1-9.3 treatment led to significant regulation of L1-dependent genes asdemonstrated for HOXA9, P3 integrin and IER 3 (FIG. 4C).

1.2.5 Analysis of Tumorigenicity in Nude Mice

Next, we investigated whether the intraperitoneal growth of SKOV3ip inmice could be inhibited by treatment with the mAbs L1-11A, L1-9.3 orL1-14.10. SKOV3ip-lacZ cells were injected into the peritoneal cavity offemale nude mice 2 days before the onset of therapy. Biweekly i.p.treatments were done using the 10 mg/kg antibody concentration. Controlmice were treated with PBS or HEA125 (anti EpCAM) as a control antibody(biweekly 10 mg/kg i.p.). In all anti-L1 mAb treatment groups, asubstantial decrease in the amount of tumor mass was visible comparedwith PBS or the control antibody HEA-125 (FIG. 5). Compared with thecontrol, all anti-L1 mAbs led to a dose-dependent reduction of i.p.tumor burden [L1-11A (10 mg/kg), −40%; L1-14.10 (10 mg/kg), −30%; L1-9.3(10 mg/kg), −60%; FIG. 5]. Tumor reduction in the group treated with theL1-9.3 (10 mg/kg) was statistically significant(P_(L1-9.3 (10 mg/kg))=0.004) compared with the PBS control. Micetreated with the HEA125 control antibody revealed no detectablereduction of SKOV3ip-lacZ i.p. tumor burden compared with thePBS-treated group (FIG. 5), although EpCAM is present on the SKOV3ipcells and HEA125 can bind to the tumor cells. No side effects or severetoxicity of L1 mAbs L-11A, L1-9.3 or L1-14.10 treatment was observedduring the whole course of treatment.

Thus, treatment with antibodies to L reduced the tumor growth SKOV3ipcells (FIG. 5) suggesting that antibodies to L1 can regulate geneexpression but also affect in vivo tumor growth.

1.2.6 Biacore Studies of the New L1 Antibodies

This study was performed by Avidex (Oxford) as described in Example 6.Table 1 summerizes these results concerning the binding kinetics of thenew L1 antibodies (ka, kd and KD).

1.2.7 Epitop-Mapping of L1-9.3 Binding Site

An important factor for the characterization of novel L1 antibodies isto examine their binding sites in L. Therefore, we constructed a varietyof L1-Fc fusion proteins covering different parts of the molecule. PCRproducts were amplified coding different length of L1 ectodomainregions. These constructs were cloned into the pig vector, and expressedas Fc-fusion proteins. After purification, products were used forWestern blot analysis. For comparing the results, we analyzed otherrecombinant L1 protein fragments (obtained from Ricardo Gouveia, Oeixas,Portugal). L1-9.3 was found to bind to first Ig domain of L1 (FIG. 6).L1-14.10 binds in the third Ig domain whereas L1-11A binds between theFN3-5 site (FIG. 6).

1.2.8 mAB L1-9.3 Blocks L1-L1 Homophilic Binding

We asked if the L1 antibodies could interfere with the homophilicbinding function of L1. To address this question, we used a celladhesion assay in which L1-transfected cells are allowed to bind toimmobilized L1. After initial coating of glass slides with a recombinantL1-Fc fusion protein, fibronectin for positive control (to which cellsbind in an integrin dependent manner) or BSA as a negative control, weincubated J558-L1 cells with L1-11A, L1-9.3 or L1-14.10 antibody. Forcontrol, we used an IgG-control, PBS or an antibody to CD24 (SWA11). ThemAb L1-9.3 could completely block the L1-L1 homophilic binding, whereasall other tested antibodies could not interfere with the homophilicbinding capacity. None of the antibodies interfered with the binding tofibronectin (data not shown).

1.3 Materials and Methods

1.3.1 Cell Lines and Cell Culture

The human ovarian carcinoma cell lines SKOV3ip (kindly provided by EllenVitetta, University of Texas, Dallas, Tex.) and OVMz were grown in DMEM(Biochrom, Berlin, Germany) with 10% FCS under cell culture conditions(5% CO₂, 95% relative humidity, 37° C.). For identification andquantification of tumor mass, the SKOV3ip cells were stably transducedwith a lacZ-encoding retroviral vector (GeneSuppressor RetroviralSystem, Biocarta, Hamburg, Germany). The chinese hamster ovary cell lineCHO stably expressing human L1 (−hL1) were established by transfectionwith superfect (Stratagene, Heidelberg, Germany) and selection for L1expression with mAb L1-11A and magnetic beads (Myltenyi Biotec, BergischGladbach, Germany) or sorting with FACS Calibur. All cells werecultivated in DMEM supplemented with 10% FCS at 37° C., 5% CO₂ and 100%humidity. Human L1 encoding plasmids and J558-L1 cells were obtainedfrom Dr. Vance Lemmon (University of Miami, Miami, Fla., USA).

1.3.2 Antibodies

HEA-125, a mouse IgG1 directed against EpCAM, was described before andbinds to all human adenocarcinomas (Moldenhauer et al., 1987).Monoclonal antibody L1-14.10 (Huszar et al., 2006), L1-9.3, L1-35.9 andL1-38.12 were obtained after immunization of mice with human L-Fcprotein comprising the ectodomain of L1 (Oleszewski et al., 1999). Goatanti-mouse IgG was affinity purified and absorbed to human serumproteins (Zymed Laboratories, Inc., San Francisco, Calif.).

1.3.3 Biochemical Analysis

SDS-PAGE and transfer of separated proteins to Immobilon membranes usingsemi-dry blotting were described before (Gutwein et al., 2000). Afterblocking with 5% skim milk in TBS or 1% BSA in TBS/0.1% Tween-20, theblots were developed with the respective primary antibody followed byperoxidase conjugated secondary antibody and ECL detection.

1.3.4 FACS Analysis

The surface staining of cells with saturating amounts of mAbs, eitherhybridoma supernatants or purified antibodies, and PE-conjugated goatantibodies to mouse Ig (Dianova, Hamburg, Germany) has been describedelsewhere (Ebeling et al., 1996). Stained cells were analyzed with aFACScan (Becton Dickinson).

1.3.5 Immunofluorescence

For immunofluorescent staining, cells were grown on coverslips, treatedfor 10 min with pervanadate and fixed for 20 min with 4%paraformaldehyde/PBS at room temperature. Cells were washed in PBS andpermeabilized with 0.1% NP-40 in PBS containing 5% goat serum for 15 minat room temperature. Cells were then incubated for 1 hour with firstantibody (phospho-specific Erk1/2). After 3 washing steps with PBS cellswere incubated 30 min in the dark to a second Alexa488-conjugated goatanti-mouse IgG. After washing the cells twice with PBS, stained cellswere mounted on glass slides and examined with an epifluorescencemicroscope (Axioplan-2; Zeiss, Oberkochem).

1.3.6 Invasion Assay

Tumor cell invasion in vitro was determined in a double-filter assay asdescribed previously in Erkell et al. (1988). Briefly, a Matrigel waslayered between two filters, a lower 5 μm pore nitrocellulose filter andan upper 8 μm pore polycarbonate filter. Following incubation of 10⁵cells with the filter sandwich for 20 h in 1 ml medium, the sandwich wasfixed and the filters separated and stained with DAPI. Cells present inthe gel on the lower filter were counted, and cell invasion wasexpressed as the ration of the cell number on the lower filter to thetotal number of cells present on both filters.

1.3.7 Quantitative PCR

For qPCR the cDNA was purified on Microspin G-50 columns (GE Healthcare,München, Germany) and quantitated by NanoDrop spectrophotometer(ND-1000. Kisker-Biotechnology, Steinfurt, Germany). Primers for qPCRwere designed with the DNA Star Program and were produced by MWG(Ebersberg, Germany). β-actin was used as an internal standard. The PCRreaction was performed with the SYBRgreen mastermix (Applied biosystems,Darmstadt, Germany).

1.3.8 Cell Binding Assay

Cell binding assays to L1-Fc or fibronectin are described in detail inOleszewski et al (JCB 2000).

1.3.9 Tumor Model and Therapy

Pathogen-free, female athymic CD1 nu/nu mice (7-9 weeks old; 20 g onaverage; Charles River) were inoculated with 5×10⁶ human lacZ-taggedovarian carcinoma cells (SKOV3ip-lacZ) into the peritoneal cavity at day0, leading to i.p. tumor formation within 5 weeks. Anti-L1 mAbs werediluted in sterile PBS to the concentration needed for treatment.Tumor-bearing mice were treated i.p. twice weekly with a 300 μL solutionof the respective dosage (10 mg/kg per application, respectively),vehicle (PBS), or Hea125 antibody control. Antibody treatments startedfrom day 3 after tumor cell injection to give the tumor cells time toattach to the inner side of the abdominal wall and the surfaces of thei.p. organs. At autopsy (day 38), ascites was sampled from all mice andthe volume was determined. All i.p. organs (including tumor mass), theabdominal wall, and the diaphragm were removed, stained withβ-galactosidase substrate (X-gal; Roche-Diagnostics, Penzberg, Germany),photographed, and weighed. The indigo blue tumor mass between theorgans, on the diaphragm and the inner site of the abdominal wall, wasremoved and weighed alone. The relative tumor burden in each mouse wascalculated by dividing tumor mass weight by total situs weight.

2. Example 2 Humanization of the Anti-L11 Murine Antibody L1_(—)9.3

In order to humanize the murine anti-L1 antibody L1_(—)9.3, the genes ofhuman v-kappa 1 (humκ1), and variable heavy chain family III (humIII)were utilised as the acceptor sequences. The numbering system usedherein for these genes is adopted from Wu and Kabat (Kabat, E. A. Wu, T.T., Perry, H M, Gottesman, K S and Foeller, C (1992) Sequences ofproteins of immunological interest, Diane Books Publishing company). Themurine L1_(—)9.3 antibody light and heavy chain amino acid sequenceswere aligned against the amino acid sequences of the humid light chainand the humIII heavy chain respectively. Two humanized L1_(—)9.3antibodies (L1_(—)9.3Hu and L1_(—)9.3Hu3) were generated by replacingthe six CDRs of the human antibody with the corresponding CDRs from themurine L1_(—)9.3 antibody.

Locations of the Six Complementarity Determining Regions (CDRs)

Kabat numbering Loop scheme LCDR1 L24--L34 LCDR2 L50--L56 LCDR3 L89--L97HCDR1 H31--H35B HCDR2 H50--H65 HCDR3 H93--H101

A number of framework residues of the murine L1_(—)9.3 antibody weretransferred to the humanized L1_(—)9.3 antibodies:

Version 1 (L1_(—)9.3Hu) humanized antibody—heavy chain residue numbers6, 23, 27, 30, 43, 49, 71, 73, 76, 78 and 94, and light chain residuenumber 100 were transferred from the murine L1_(—)9.3 antibody and lightchain residue number 73 was replaced with the corresponding (Phe) foundat this position in the human REI antibody light chain.

Version 2 (L1_(—)9.3Hu3) humanized antibody—heavy chain residue numbers6, 23, 27, 30, 71, 73, and 94, and light chain residue number 100 weretransferred from the murine L1_(—)9.3 antibody.

DNA sequences encoding single-chain variable fragment (scFv) analoguesof the murine L1_(—)9.3 antibody and the two humanised versions of thisantibody (L1_(—)9.3Hu, and L1_(—)9.3Hu3) for expression in E. coli. werethen generated. All of these scFvs contain the same linker(TSGPGDGGKGGPGKGPGGEGTKGTGPGG (SEQ ID NO: 12)). The scFv genes weresynthesized by GeneArt AG, Germany.

The antibody light chain and heavy chain DNA sequences used to constructthe humanized antibodies are provided in FIGS. 8 a and 8 b respectively.

FIG. 9 a-9 c provide the amino acid sequences of the murine L1_(—)9.3scFv and the humanized L1_(—)9.3Hu and L1_(—)9.3Hu3 scFvs respectively.

3. Example 3 Cloning of DNA Encoding the L1_(—)9.3, L1_(—)9.3Hu andL_(—)9.3Hu3 scFvs into E. coli Periplasmic Expression Vectors andTransformation of E. coli with these Vectors

Periplasmic expressed of scFvs is beneficial for a number of reasons.Firstly, such scFvs leak into the bacterial supernatant and from therecan conveniently be assayed for binding to their cognate antigen (The L1cancer antigen in this case). Secondly, periplasmic expression allowsfor purification of soluble active scFvs.

The DNA sequences encoding the L_(—)9.3, L1-9.3Hu and L1_(—)9.3Hu3 scFvsas synthesized by GeneArt AG, Germany were not supplied in an E. coliperiplasmic expression vector. Therefore, these DNA sequences wherecloned into an E. coli periplasmic expression vector using the followingmethods.

The DNA encoding the synthesized scFvs were PCR rescued with thefollowing primer pairs using standard PCR conditions and reagents:

scFv Primer pair L1_9.3 Yol811 and Yol812 L1-9.3Hu Yol813 and Yol814L1_9.3Hu3 Yol813 and Yol814

Yol811              (SEQ ID NO: 13) AGCCGGCCATGGCCGATATTCAGATGACCCAGACYol812              (SEQ ID NO: 14)TCTATGCAGCGGCGGCACCGCCGCTGCTCACGGTAACGCTGYol813              (SEQ ID NO: 15) AGCCGGCCATGGCCGATATTCAGATGACCCAGAGYol814              (SEQ ID NO: 16)TCTATGCAGCGGCCGCACCGCCGCTGCTCACGGTAACCAGGGTG

The PCR products were run on a 1.6% agarose gel and bands of the correctsize excised and purified. The PCR products were double digested withNco1 and Not1 restriction enzymes under standard conditions followed byre-purification. The PCR products were ligated into an IPTG inducibleperiplasmic expression vector which contained:

-   -   a pelB leader sequence to direct the encoded polypeptides to the        periplasm where this leader sequence is then cleaved off    -   Nco1/Not1 cloning sites    -   the human antibody kappa chain constant region

The ligated vectors were transformed into E. coli TG1 cells and platedon of 2×TY agar (Bacto Trypton 16 g/L, yeast extract 10 g/L, 15 g/Lbactoagar and NaCl 5 g/L) supplemented with 100 μg/ml ampicillin and 2%glucose. The DNA and amino acid sequences of the expressed portions ofL1_(—)9.3, L1-9.3Hu and L1_(—)9.3Hu3 scFv constructs are shown in FIGS.10 a, 10 b and 10 c respectively.

4. Example 4 Expression of L1_(—)9.3, L1-9.3Hu and L1_(—)9.3Hu3Single-Chain Antibodies in E. coli

The polypeptides expressed by these vectors include the human antibody ckappa constant region fused to the C termini of the scFvs. These c kappaconstant chain containing constructs are referred to herein as singlechain antibodies.

Eight E. coli clones for each single chain antibody construct,L1_(—)9.3, L1_(—)9.3Hu, and L1_(—)9Hu3, (24 clones in total) were pickedinto separate wells of a 96 well plate containing 300 μl of 2×TY (BactoTrypton 16 g/L, yeast extract 10 g/L and NaCl 5 g/L) supplemented with100 μg/ml ampicillin and 2% glucose. Each well has a 1 ml volume. Thecultures were grown with shaking (200 rpm) at 37° C. until the culturesreached an OD₆₀₀ of approximately 0.5. The 96 well plates were then spundown at 3200 rpm for 10 min and the supernatant was aspirated anddiscarded. The bacterial pellets were resuspended in fresh 2×TY 400 μlsupplemented with 100 μg/ml ampicillin and 1 mM IPTG to induceexpression of the single chain antibodies. The cultures were shaken at200 rpm overnight at 25° C.

The following day the 96 well plate was spun down at 3200 rpm for 10 minto pellet the cells. The supernatant containing the expressed L1 singlechain antibodies was kept for ELISA analysis.

5. Example 5 ELISA Assay of Binding of the L1_(—)9.3, L1-93Hu andL19.3Hu3 scFvs to Human L1 Cancer Antigen

This ELISA assay was carried out in order to confirm that thehumanisation process had not lead to a loss of antibody binding to theL1 cancer antigen and to identify which of the clones picked correctlyexpressed the single chain antibody constructs.

Three rows of a 96 well plate were coated with 100 μl L1 antigencomprising the extracellolar domain of the L1 protein fused to an Fcfragment (5 μg/ml) in PBS for 1 hr at room temperature. A further threerows were coated with streptavidin (5 μg/ml) in PBS as a control.

The wells were washed three times with 370 μl of PBS and blocked with 3%milk powder in PBS for 1 hr at room temperature.

50 μl of each overnight bacterial supernatant was mixed with 50 μl of 6%milk powder in PBS for 1 hour.

The blocked ELISA plate was washed twice with PBS as described above andthe blocked supernatants containing single chain antibody were added andincubated for 1 hp at room temperature.

The 96 well plate was washed four times with PBS 0.1% tween followed bythe addition of 100 μl of anti-human kappa light chains bound and freeantibody HRP conjugate (Sigma A7164) 1:5000 dilution in PBS 1% BSA. Theconjugate was incubated for 1 hr at room temperature followed by fivewashes with PBS 0.1% tween.

The ELISA was developed by the addition of TMB 2-Component MicrowellPeroxidase Substrate Kit (Kirkegaard and Perry Laboratories Inc., USA)according to the manufacturer's protocol. An image of the ELISA plate isshown in FIG. 4. At least four L1 binding clones have been observed foreach of three single chain antibody versions. These L1 binding singlechain antibody clones do not bind to streptavidin.

FIG. 11 shows the binding of the L1_(—)9.3, L1-9.3Hu and L1_(—)9.3Hu3scFvs to the human L1 cancer antigen. Rows A, B and C are coated with L1and rows D, E and F are coated with streptavidin. The blue colour in thewells indicates binding of the individual scFv to the L1 on the plate.The lack of colour in the streptavidin coated rows shows that the singlechain antibodies are specifically binding to L1.

6. Example 6 Determination of Binding Affinity

Mouse antibody L1-9.3 and humanised antibody L1-hu3 were assayed byBiacore analysis (Biacore AB, Uppsala, Sweden) to determine bindingkinetics.

A BIAcore CM5 sensor chip was activated with EDC/NHS and purifiedrecombinant L1-Fc extracellular fragment (515 μg/ml in PBS) was coupledto the CM5 sensor chip to between 200 and 3000 RU. The remaining activesites were blocked by ethanolamine/HCl. Antibody binding was measured byadding antibody at concentrations from 6 to 3333 nM at a flow rate of 10ul/min using the Kinject function. The chip was regenerated with 10 mMGlycine pH 2.0 with 500 mM NaCl to remove the bound antibodies.

The binding curves were fit to a Langmuir binding model usingBIAevaluation software (Biacore AB, Uppsala, Sweden). Determined KDvalues are shown in Table 2.

TABLE 2 Table 2: The humanized variant L1-hu3 displays a similar hightarget affinity as the parent antibody L1-9.3. Antibody L1-9.3 L1-hu3 Ka[1/Ms] 2.6 × 10⁵ 8.0 × 10⁵ Kd [1/s] 2.2 × 10−⁵ 6.5 × 10−⁵ KD [M] 8.5 ×10−¹¹ 8.1 × 10−¹¹

7. Example 7 Antibody Binding to PBMCs and Cancer Cells

PBMC were obtained by density gradient centrifugation from EDTA wholeblood of healthy human donors. Cultured OVMZ tumor cells were harvestedby trypsination. 1×10⁵ cells/well (75 μl) were seeded into FACS tubes.Dilutions of L1-9.3 mAb were prepared in culture medium with 10 mM EDTAand 75 μl/well of L1-mAb dilution were added, to PBMCs and OVMZ cells toresult in final concentrations between 6.6×10⁻¹³ to 6.6×10⁻⁸ Mol.Subsequently cells were incubated over night (˜24 h) at 37° C./5% CO₂ inan incubator. Cells were washed directly in FACS tubes using 2 ml ofFACS buffer followed by centrifugation at 300 g/5 min/4° C. Thesupernatant was removed by pipetting. For staining, a PE-labelled donkeyanti-mouse secondary antibody (Dako) was added at a volume of 150μl/well followed by incubation for 30 min at 4° C. Washing steps wererepeated as above and cells were fixed in 200 μl PBS/1% formaldehyde.Sample mean fluorescence was then measured by FACS analysis.

As shown in FIG. 13, L1-9.3 mAb displays a strongly reduced affinity toL1 on PBMC compared to tumor L1. L1-9.3 binding to PBMC was detected inthe nanomolar range (dashed line), while binding to tumor cells could beobserved at picomolar concentrations (solid line). B) The dissociationconstants K_(D) were estimated from the regression curves using theconcentration at half-maximal binding. K_(D) of L1-9.3 on PBMC was atleast 400-fold lower than on tumor cells.

8. Example 8 Determination of Cytokine Release

PBMC were obtained by density gradient centrifugation from citrate wholeblood of healthy human donors. Cells were resuspended in RPMI 1640/5%human serum/5 ml NEAA/5 ml L-Glutamin/5 ml Natrium-Pyruvat. 1×10⁵ cellsper 100 μl were seeded in round bottom 96 well plates. In a second step,100 μl medium containing LPS (10 ng/ml), L1-9.3 mAb (20 μg/ml), OKT3 mAB(ebioscience) (75 ng/ml) or Ionomycin/PMA (1 μg/ml/5 ng/ml) were addedin triplicates followed by an incubation for 24 h at 37° C., 5% CO₂. Asnegative control, untreated PBMC were used. After 24 h, levels of thecytokines interferone-gamma and tumor necrosis factor were measured byFACS analysis using the CBA-Cytokin-Flex-Sets (BD) according tomanufacturers information.

The resulting cytokine levels are depicted in FIG. 14. In contrast toOKT3 mAB, Ionomycin/PMA, and LPS, L1-9.3 did not significantly increasethe TNF or IFN-gamma release by PBMCs.

9. Example 9 T-Cell Proliferation Assay

PBMC were obtained by density gradient centrifugation from citrate wholeblood of two healthy human donors. 1×10⁵ cells per well were seeded inflat bottom 96 well plates. In a second step, 100 μl medium containingeither L1-9.3 mAb (20 μg/ml) and OKT3 (ebioscience, 75 ng/ml) or L1-9.3mAb (20 μg/ml) or OKT3 (75 ng/ml) was added in triplicates. After 1 h,the latter two were supplemented with OKT3 or L1-9.3, respectively. Toexclude any antibody related activation, PBMC with or without L1-9.3were incubated in absence of OKT3. Following an incubation for 24 h at37° C., 5% CO₂ T cell proliferation was assessed using a BrdUincorporation assay (Roche) according to manufacturers information.

It can be concluded from the results shown in FIG. 15, that L1-9.3 mAbdoes neither induce T-cell proliferation or inhibit OKT3 induced T-cellproliferation.

10. Example 10 Glycosylation Dependency of Antibody Binding

2×10⁶ SKOV3ip cells were seeded in a 10 cm petri dish and incubated for24 h at 37° C., 5% CO₂. After 24 h, cells were washed with PBS and lysedwith 500 μl M-PER reagent (Pierce) according to the protocol describedin the Seize Classic Mammalian Immunoprecipitation Kit (Pierce). SkOv3ipcell lysate were deglycosylated as described in the EnzymaticCarboRelease Kit (QA_Bio). Briefly, 2.5 μl denaturation solution wasadded to 35 μl of cell lysate. The sample was incubated in a thermoblockat 100° C. for 5 min and then chilled on ice. Finally 2.5 μl Triton-Xand 1 μl of each glycosidase contained in the Enzymatic CarboRelease Kit(QA_Bio) (PGNase F, O-Glycosidase, Sialidase, β-Galactosidase,Glucoaminidase) were added according to manufacturers protocol followedby an incubation at 37° C. for 3 h. Glycosylated and deglycosylated weresubjected to SDS PAGE and subsequent Western blotting. Western blotswere incubated with different L1 antibodies in dependence of theirstaining performance. Concentrations of 1 μg/ml (9.3, 11A and 14.10), 5μg/ml (35.9) or 10 μg/ml (OV52.24, OV543.18, 38.12, OV549.20) were used.L1 antibody binding to western blot was detected with HRP-labeledanti-mouse antibody (Dianova).

As shown in FIG. 16, the tested anti L1 antibodies can be divided intothree classes in respect to their glycosylation-dependency: First class(unaffected by glycosylation): L1-9.3. Second class (binding in WB wasnegatively affected by deglycosylation): 11A, 14.10, OV52.24 andOV549.20. Third class (binding in WB was positively affected bydeglycosylation): 35.9 and 38.12.

11. Example 11 Biodistribution of L1-9.3 in Rabbit

A female rabbit (White Himalayan) was twice injected with L1-9.3 (0 h,24 h) via the intravenous application route at a dose of 10 mg/kg. 1control animal received a comparable volume of PBS. Animals werenecropsied 72 h after the first application. Organs were fixed in 4%buffered formalin and embedded in paraffin. Histological slides wereprepared and immunohistochemistry was performed. Tissue sections of theL1-9.3-treated and control animal were stained with an anti-mouseantibody to detect binding of L1-9.3 after intravenous application.Signals were visualized by DAB (Sigma). Two different detection systems,conventional Avidin/Biotin Complex method or tyramide signalamplification system CSA II method (Dako) were used, which allowed roughestimation of the amount of in vivo bound L1-9.3. The conventionalAvidin/Biotin Complex method (Vector Laboratories) is able to detectL1-9.3 concentrations of 50 ng/ml or higher, while the biotin-freetyramide signal amplification system CSA II (Dako) has a detection limitof 5 ng/ml. To determine the L1 expression pattern, tissues of thecontrol animal were incubated with primary antibody L1-9.3 and with thedetection antibody. For ABC method a biotinylated anti-mouse antibody(Dianova, dilution 1:3000) was used as detection antibody, for CSAmethod was performed according to manufacturers protocoll.

FIG. 17 shows the in vivo binding of intravenously applied L1-9.3 tocollecting ducts of the kidney. In vivo binding was only detectableusing the amplification system CSA (FIG. 17A), while by using theconventional ABC-method, no signal was visible (FIG. 17B). Hence, L1-9.3was detected in a range of 30-300 pmol in the tissue (L1-9.3concentration is presumably higher than 5 ng/ml and below 50 ng/ml).Negative control did not show staining, thus, unspecific staining can beexcluded (FIG. 17C). The staining pattern of in vivo bound L1-9.3 (FIG.17A) corresponds to the L1 expression pattern in the kidney whendirectly staining tissue sections with L1-9.3 (FIG. 17D). It can beconcluded that intravenously administered L1-9.3 antibody is able toextravasate to peripheral tissue.

12. Example 12 Function of humanized forms of L1 9.3 mAb in Nude Mice

We investigated whether the humanized form of the mAb L1 9.3 could alsoinhibit the tumor growth of ovarian carcinoma in vivo. First we analysedthe binding of the two humanized forms of L1 9.3 to the selected cellline. Therefore, flow cytometry was performed on SKOV3ip pcDNA3.1Luciferase cells. (FIG. 18). Both mAbs showed strong binding to thetumor cell line, and gave similar binding results as the native L1 9.3mAb.

SKOV3ip pcDNA3.1 Luciferase cells were injected into immunodeficientmice 24 h before starting the therapy. Humanized antibodies (300 μg) orPBS were injected three times per week intraperitoneally. To detect thetumor growth in vivo, mice were imaged once weekly using the XenogenTVIS 200 System. Mice were anesthetised and injected with Luciferin D,followed by detecting the light emission which is produced duringluciferase activity of the tumor cells. During the time course wedetected a slower tumor growth in the group of mice treated withhumanized mAb compared to the control. At day 33 the last imaging datawere taken. Imaging results gave a decreased tumor volume of around 80%using the hu3 mAb and approximately of 50% for chiL1 9.3. Both resultswere strongly significant (FIG. 19). After 36 days mice were sacrificedand tumor mass has determined. In both humanized anti-L1 mAbs treatedgroups a substantial decreased tumor mass was measured compared to thePBS group (FIGS. 20 (A, B)).

13. Example 13

Abolishment of chemoresistance by treatment with anti L1CAM monoclonalantibody 9.3 was tested as described in WO 2008/046529, Example 3 (seealso FIG. 17e of WO 2008/046529). The results are shown in FIGS. 21 and22. It could be demonstrated that the monoclonal antibody 9.3 abolisheschemoresistance. Its effect seems to be stronger than those of theantibody 11A tested in WO 2008/046529.

TABLE 1 Western mAb FACS blot IP L1-Fc Invasion phospho-Erk ka (1/Ms) kd(1/s) KD (M) tumor growth L1-9.3 +++ +++ +++ +++ −60% −50% 2.6E+052.2E−05 8.5E−11 −60% L1-11A +++ +++ +++ +++ −50% −40% 1.0E+05 4.0E−064.0E−11 −40% L1-14.10 + ++ + +++ −40% −40% 1.4E+04 1.0E−06 7.1E−11 −30%L1-38.12 + +++ + +++ 0 0 3.7E+04 2.0E−06 5.4E−11 L1-35.9 + +++ + +++ 0 04.0E+04 1.2E−05 3.0E−10 L1-N15.17 ++ − ++ ++ 0 0 5.3E+04 1.0E−03 1.9E−08L1-1D12.22 − − + ++ 0 −20% 2.3E+04 1.0E−04 4.3E−09 L1-1D17.3 − − + ++ 00 2.3E+04 1.0E−04 4.3E−09 L1-1D64.8 − +++ + +++ 0 0 8.5E+04 1.5E−041.8E−09 L1-1D74.8 − +++ + +++ −10% 0 3.0E+04 2.0E−03 6.7E−08

The invention claimed is:
 1. A method for treating tumor cells or atumor, wherein an antibody or an antigen-binding fragment thereofselected from the group consisting of i) an anti-L1CAM monoclonalantibody which binds to the same L1CAM epitope recognized by themonoclonal antibody 9.3, produced by the hybridoma cell deposited underDSMZ ACC2841, ii) an anti-L1CAM monoclonal antibody, wherein its sixcomplementarity determining regions (CDRs) comprise the followingsequences: RASQDISNYLN (SEQ ID NO: 1), YTSRLHS (SEQ ID NO: 2), QQGNTLPWT(SEQ ID NO: 3), RYWML (SEQ ID NO: 4), EINPRNDRTNYNEKFKT (SEQ ID NO: 5),and GGGYAMDY (SEQ ID NO: 6) iii) a monoclonal antibody, produced by thehybridoma cell deposited under DSMZ ACC2841; and iv) an antibody orantigen-binding fragment thereof comprising the following sequences:RASQDISNYLN (SEQ ID NO: 1), YTSRLHS (SEQ ID NO: 2), QQGNTLPWT (SEQ IDNO: 3), RYWML (SEQ ID NO: 4), EINPRNDRTNYNEKFKT (SEQ ID NO: 5), andGGGYAMDY (SEQ ID NO: 6) is administered to a subject in an effectiveamount to treat said tumor cells or tumor.
 2. The method of claim 1,wherein the antibody or antigen-binding fragment thereof binds to theepitope that is within the first immunoglobulin-like domain of L1CAM. 3.The method of claim 1, wherein the antibody is a humanized antibody. 4.The method of claim 1, wherein the antibody is a humanized antibodyhaving non-human CDRs and human framework region (FR).
 5. The method ofclaim 1, wherein the antibody is a humanized antibody comprising (i) SEQID NOs: 20 and 24; or (ii) SEQ ID NOs: 21 and
 25. 6. The method of claim1, wherein the antibody or antigen-binding fragment thereof is selectedfrom the group consisting of single chain antibodies (scFv), multimersof scFv, diabodies, triabodies, tetrabodies, Fab, tandabs, flexibodies,bispecific antibodies, and chimeric antibodies.
 7. The method of claim1, wherein the antibody or antigen-binding fragment thereof is linked toan active substance.
 8. The method of claim 1 for sensitizing tumorcells in a patient for the treatment with a chemotherapeutic drug orwith radiotherapy.
 9. The method of claim 1 for sensitizing tumor cellsin a patient for the treatment with a chemotherapeutic drug or withradiotherapy, wherein the cells are at least partially resistant to thetreatment with said chemotherapeutic drug or to radiotherapy.
 10. Themethod of claim 1 for sensitizing tumor cells in a patient for thetreatment with a chemotherapeutic drug or with radiotherapy, whereinafter the sensitization with the antibody or antigen-binding fragmentthereof the patient is further treated with said chemotherapeutic drugor with radiotherapy.
 11. The method of claim 1 for the treatment of atumor in a patient previously treated with a chemotherapeutic drug orwith radiotherapy.
 12. The method of claim 1 for the treatment of atumor in a patient at least partially resistant to treatment with agiven chemotherapeutic drug or with radiotherapy.
 13. The method ofclaim 1, wherein the antibody or antigen-binding fragment thereof isadministered in combination with a chemotherapeutic drug or withradiotherapy.
 14. The method of claim 1, wherein the antibody orantigen-binding fragment thereof is administered in combination with achemotherapeutic drug or with radiotherapy, wherein the chemotherapeuticdrug or the radiotherapy is administered prior to the antibody orantigen-binding fragment thereof.
 15. The method of claim 1, wherein thetumor cells or the tumor are of a type selected from the groupconsisting of astrocytoma, oligodendroglioma, meningioma, neurofibroma,glioblastoma, ependymoma, Schwannoma, neurofibrosarcoma,medulloblastoma, melanoma, pancreatic cancer, prostate carcinoma, headand neck cancer, breast cancer, lung cancer, ovarian cancer, endometrialcancer, renal cancer, neuroblastomas, squamous carcinomas,medulloblastomas, hepatoma, colon cancer, mesothelioma and epidermoidcarcinoma.
 16. The method of claim 1, wherein the tumor is an epithelialtumor.
 17. The method of claim 1 for sensitizing tumor cells in apatient for the treatment with a chemotherapeutic drug or withradiotherapy, wherein the chemotherapeutic drug is a DNA damaging agent.18. The method of claim 17 for sensitizing tumor cells in a patient forthe treatment with a chemotherapeutic drug or with radiotherapy, whereinthe radiotherapy is selected from the group consisting of X-rayradiation, UV-radiation, γ-irradiation, α- or β-irradiation, andmicrowaves.
 19. The method of claim 1, wherein the antibody orantigen-binding fragment thereof is an anti-L1CAM monoclonal antibodyhaving the same capacity to inhibit tumor growth as the monoclonalantibody 9.3, produced by the hybridoma cell deposited under DSMZACC2841.
 20. The method of claim 7, wherein the active substance isselected from the group consisting of a toxin, a cytotoxin, ananoparticle, and a radionuclide.
 21. The method of claim 17, whereinthe DNA damaging agent is selected from the group consisting ofactinomycin-D, mitomycin C, cisplatin, doxorubicin, etoposide,verapamil, podophyllotoxin, 5-FU, taxans, paclitaxel, and carboplatin.22. The method of claim 16, wherein the epithelial tumor is pancreaticcancer, colon cancer, ovarian cancer or endometrial cancer.